Monomeric site-specific nucleases for genome editing

Kleinstiver, Benjamin P.; Wolfs, Jason M.; Kolaczyk, Tomasz; Roberts, Alanna K.; Hu, Sherry; Edgell, David R.

doi:10.1073/pnas.1117984109

Cited by 50 publications

(66 citation statements)

References 43 publications

(67 reference statements)

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“…The I-TevI nuclease and linker domains have successfully been fused to four different DNA-binding architectures that are used in genome editing: zinc fingers (27), TALEs (29), meganucleases (30), and, as reported here, Cas9. An on-going debate in the genomeediting field centers on the ease of use (targeting range) versus specificity (off-target effects) of the various reagents, particularly for common laboratory manipulations of cell lines or model organisms.…”

Section: Discussionmentioning

confidence: 99%

“…Subsequent repair would effectively delete the majority of the target site, preventing further cleavage. We previously created a dual nuclease termed MegaTev, a fusion of I-TevI to LAGLIDADG homing endonucleases (or meganuclease) (27,34,35). We showed that MegaTevs efficiently excises a 30-bp fragment from model DNA substrates in HEK293 cells (30).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies revealed that the length of the DNA spacer is crucial for I-TevI cleavage function, with lengths of 14-19 bp supporting activity in various chimeric contexts (27,29,30). We coexpressed TevCas9 with a C-terminal histidine tag and a gRNA targeting a site in the retinoic acid receptor alpha gene (RARA.233, numbered according to start of target site in the RARA cDNA) from the same plasmid in Escherichia coli.…”

Section: Significancementioning

confidence: 99%

“…To create a TevCas9 RNA-guided dual nuclease, we fused the I-TevI nuclease domain (residues 1-92) and a portion of the linker domain (residues 92-169) to the N terminus of SpCas9 nuclease derived from the Streptococcus pyogenes type II CRISPR system (27,28) (Fig. 1A).…”

Section: Fusion Of I-tevi To Cas9mentioning

confidence: 99%

“…We created a dual nuclease that introduces two noncompatible DNA breaks at a target site such that the majority of the target site is deleted, preventing regeneration of the target site and continued cleavage. We fused the monomeric nuclease and linker domains from the I-TevI homing endonuclease to Cas9 (27,28), creating an RNA-guided TevCas9 dual nuclease that functions robustly in HEK293 cells at endogenous target sites. TevCas9 generates defined length deletions of 33-36 bp at high frequencies with minimal DNA end processing compared with Cas9.…”

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Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Wolfs

Hamilton

Lant

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33-to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.CRISPR/Cas9 | genome editing | NHEJ | I-TevI homing endonuclease G enome editing with engineered nucleases has revolutionized the targeted manipulation of the genomes of organisms ranging from bacteria to mammals (1). Zinc finger nucleases (ZFNs) (2), transcription-like effector nucleases (TALENs) (3), MegaTALs (fusion of a LAGLIDADG homing endonuclease and TALE domain) (4-6), and nucleases based on the CRISPR-associated protein 9 (Cas9) all represent programmable genome-editing nucleases that have successfully been used to introduce targeted changes in genomes (7-11). One of the most common applications of genome-editing nucleases is gene knockouts that are performed in the absence of an exogenously added repair template (12). In the case of Cas9, the blunt DNA ends introduced at DNA cleavage are substrates for error-free repair by the classical nonhomologous endjoining repair (c-NHEJ) pathway (13), regenerating the target site for recleavage by the nuclease. This cycle of cleavage, ligation, and target site regeneration is perturbed when the double-strand break (DSB) is sufficiently modified by exonucleolytic processing by c-NHEJ, or by the alternative NHEJ pathway (alt-NHEJ), to prevent cleavage by the nuclease (14-18). Imprecise repair by either of the NHEJ pathways generates the characteristic spectrum of heterogeneous length insertions or deletions (indels) centered around the break site (19,20). The heterogeneous distribution of indels, and the fact that not all indels generate gene knoc...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Fusion Of I-tevi To Cas9mentioning

confidence: 99%

mentioning

confidence: 99%

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Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Wolfs

Hamilton

Lant

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondrial Dna Repair and Genome Evolution

Christensen¹

2017

Annual Plant Reviews, Volume 50

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MitochondrialDNARepair and Genome Evolution

Christensen

2018

Annual Plant Reviews Online

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In plants, replicating the mitochondrial genome is probably a very minor expenditure of energy compared to the other costly activities of the cell, including replicating the much larger nuclear genome, transcription, protein synthesis and active transport. This chapter discusses the angiosperm mitochondrial genomes. The mutational burden hypothesis (MBH) explains an inverse correlation between mutation rate and genome size. The types of DNA damage that occur in mitochondria, the available mechanisms of repair, and selection on the repaired DNA products is taken into account in understanding the patterns of plant mitochondrial genome evolution. Analysis of the evolutionary patterns seen in plant mitochondrial genomes led to hypotheses of what mechanisms of DNA repair are available in plant mitochondria. The mechanism of repair ensures accurate inheritance of genes and has the side‐effect of allowing duplications, rearrangements and the accumulation of junk DNA.

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Monomeric site-specific nucleases for genome editing

Cited by 50 publications

References 43 publications

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Mitochondrial Dna Repair and Genome Evolution

MitochondrialDNARepair and Genome Evolution

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